Heme oxygenase-1 fused to a TAT peptide transduces and protects pancreatic β-cells
Section snippets
Cloning and related techniques
The recombinant TAT/PTD anti-apoptotic protein construct was generated by inserting the DNA coding region of murine heme oxygenase-1 (HO-1) (produced by Robert Oliver University of Miami) in the EcoRI site of the pTAT expression vector (kind gift from Dr. Steven Dowdy from USDC).
Protein generation and purification
The isolation and purification of TAT/PTD–HO-1 and HO-1 (no TAT) were done as previously described [11] with some modifications in the washing steps: briefly, the Ni–NTA column, with bacterial extract, was sequentially
Generation and purification of recombinant TAT/PTD–HO-1 fusion protein
To generate a TAT/PTD–HO-1 recombinant protein we inserted the coding region of the murine heme oxygenase gene in-frame with the 11-amino-acid PTD of the HIV/TAT protein in the pTAT-HA expression vector. Bl 21 Escherichia coli was used to produce the recombinant TAT/PTD–HO-1 protein. A green coloration of the bacterial culture was consistently observed, suggesting that the recombinant TAT/PTD–HO-1 metabolized the heme from E. coli into biliverdin, that accumulated due to the lack of biliverdin
Conclusions
Our data show that transduction of insulin producing cells with a recombinant HO-1 protein fused to the TAT/PTD confers cytoprotection against TNF-α-cytotoxicity and cell death during culture. Transduction with TAT/PTD–HO-1 protein might be useful to confer transient cytoprotection and therefore enhance the viability of transplantable islets. Prolonged culture of viable islets could help develop immunosuppressive regimes for recipients prior to islet transplantation.
Acknowledgments
This work was supported by a grant from the National Institutes of Health (DK-59993) awarded to R.L.P., the Diabetes Research Institute Foundation (Hollywood, FL), and the Foundation for Diabetes Research.
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